Isolation of Total RNA from just about anything

Wed Jun 29 18:13:04 EST 1994

In article <2us1uv$quh at> acc4636 at (Anthony C.) writes:
>From: acc4636 at (Anthony C.)
>Subject: Re: Isolation of Total RNA from just about anything
>Date: 29 Jun 1994 09:54:23 -0500

>In article <2ulsl3$esu at>,
>Winoto lab <lab_winoto at> wrote:
>>RNase-All total RNA Isolation                                          Astar Winoto
>>                                                                               Paul W. Diaz
>>                                                                               Elicia Penuel
>[Deleted RNA extraction protocol]
>I am using essentially the same protocol (current protocols in molecular
>biology) and its good to see that I can probably leave out one of the
>precipitation steps with good results.  I can typically get about 30
>micrograms of total RNA from 5 million cells and an A260/A280 of about
>1.8-2.0.  My problem is that although I am reading RNA in the spec I
>simply cannot get the RNA to run on a gel.  I am using the following 
>recipe :

>11 ul of RNA (10 ug!!)
>9 ul of formaldehyde
>5 ul 10X MOPS
>25 ul formamide
>5 ul of premade gel loading buffer from SIGMA

>The gel is

>.5 grams of wide range agarose from SIGMA
>36 ml of depc treated double distilled water
>5 ml of 10X MOPS
>9 ml of formaldehyde
>5 ul of 500 ug/ml EtBr

>The gels are a little too soft (pouring buffer on sometimes rips the
>wells) so Im going to halve the formaldehyde today.  The 5 ul of EtBr
>is a little too bright so Im going to use 2 ul.  The MOPS is a 10X powder
>from sigma that you just add 1 liter of water to (minimize the things that
>can go wrong :) ).

>The problem is that I cant get my isolated RNA or an RNA marker
>(RNA marker III from boehringer mannheim, .3-1.6 bp approximately) to
>show up on the gel.  At this point I dont care if the prep is partially
>digested with RNAses.. I just want the smear!  If you have any suggestions
>let me know!  Thanks a lot. (Someone suggested I use a first strand 
>cDNA synthesis kit to make cDNA, then run that out hehe..)  Oh.. the reason
>Im isolating RNA is I eventually hope to purify the mRNA and make a 
>cDNA library.  Im quite amazed that I cant get the gel to work.  One last
>thing is that I want to know how critical the percent of formamide and
>formaldehyde in the sample solution is.  Sometimes I use a larger
>volume of RNA (say 30ul) how which dilutes the formamide and formaldehyde.
>I know they are supposed to remove the RNA secondary structure, but at
>this point Im not planning to do any hybridization so the secondary structure
>isnt too much of a concern.  Thanks in advance!

           Do you destain your gels after electrophoresis? EtBr stained RNA is 
not readily visible in the presence of formaldehyde which should be removed by 
3-4 consecutive washes with DEPC treated water over a period of about an hour. 
Sometimes even longer is required. Also, my RNA gels seem clearer if I stain 
in 0.2 ug/ml EtBr after electrophoresis.
            If all you want to do is check for the presence of or quality of 
your RNA then run a non-denaturing gel i.e just dissolve the RNA in water and 
run on a 1x TBE-agarose gel without formaldehyde. However, you will not be 
able to run your RNA markers in a non denaturing system as they will probably 
have a high degree of secondary structure and therefore will not give accurate 
size estimations.
            I hope this is of some assistance to you.

David Glover.

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